Lipid disulfide prodrugs and uses related thereto

ABSTRACT

This disclosure relates to lipid disulfide prodrugs and in particular to lipid disulfide phosphodiester nucleosides and derivatives thereof, pharmaceutical compositions, and uses related thereto. According to one embodiment of the disclosure there is provided a compound of Formula I,or pharmaceutically acceptable salts or derivatives thereof, wherein substituents are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/035,164filed Jul. 13, 2018, which claims the benefit of U.S. ProvisionalApplication No. 62/532,013 filed Jul. 13, 2017. The entirety of each ofthese applications is hereby incorporated by reference for all purposes.

BACKGROUND

Many drug candidates and natural products are flanked with structuralfeatures such as carboxylic acids, amines, dianionic phosphates andpolyhydroxylated aromatic rings, etc. that may limit their therapeuticpotential in vivo. Such features often require temporary protection witha prodrug to improve the absorption, distribution, metabolism andexcretion (ADME) properties of a pharmacologically active compoundwithin the body.

One example is Tenofovir (TFV), which is an acyclic nucleoside withanti-viral activity against human immunodeficiency virus (HIV),hepatitis B virus (HBV), and herpes simplex type-2 virus (HSV-2). TFVstructurally resembles 2′,3′-dideoxyadenosine which lacks the requisite3′ hydroxyl moiety necessary for DNA polymerization and triggersobligate chain termination upon incorporation of tenofovir diphosphate(TFVdpp) into the growing viral DNA strand. A common structural featureamongst acyclic nucleosides (e.g. TFV, adefovir, cidofovir, ganciclovir,etc.) is a catabolically stable phosphonate linkage that permanentlyaffixes the phosphonate to the acyclic sugar linker and nucleobase. Thisserves to prevent undesirable chemical and enzymatic hydrolysis andbypasses the initial phosphorylation to the monophosphate, which is thekinetic bottleneck during the conversion of conventional nucleosides totheir active triphosphate (De Clercq et al., Nature reviews. Drugdiscovery, 2005, 4, 928).

The dianionic character of TFV and other acyclic nucleosides atphysiological pH restricts diffusion across the plasma membraneresulting in rapid renal clearance and depreciated bioavailability andantiviral activity. When orally administered to mice, thebioavailability of TFV is approximately 2% and that of adefovir has beenreported to be <1% in monkeys and 8-11% in rats (see Kearney et al., J.Clin. Pharmacokinet., 2004, 43, 595, Balzarini et al. AIDS, 1991, 5, 21.Starrett et al., J. Med. Chem., 1994, 37, 1857). These undesirableproperties can be ameliorated by masking the anionic phosphonic acidwith various prodrugs that alter the pharmacokinetic profile of theparent nucleoside, enhance cellular permeability, and improvebioavailability. Several eclectic prodrug strategies have been developedfor this purpose.

The clinically-approved prodrug formulation of TFV is tenofovirdisoproxil fumarate (TDF), manufactured by Gilead Sciences under thetrade name Viread®, which features two isopropyloxymethyl carbonatemasking units esterified to the phosphonate that relies on anesterase-activated cleavage mechanism to liberate TFV followingsuccessful delivery to the target tissue. The installation of twoisopropyl carbonate esters increases the oral bioavailability of TFV to25%, dramatically enhances tissue distribution and improves biologicalstability. However, the ubiquitous distribution of esterases renders asignificant fraction of TDF susceptible to premature hydrolysisresulting in systemic exposure to TFV, a known nephrotoxin, potentiallycausing undesirable side effects (see Karras et al. Clinical infectiousdiseases: an official publication of the Infectious Diseases Society ofAmerica, 2003, 36, 1070). Continuous administration of TDF has beenreported to induce lactic acidosis, Fanconi syndrome, acute renalfailure, and bone loss, all of which stem from mitochondrial toxicity(see Fernandez et al., AIDS Res. Treat.; 2011: 354908 and Coca &Perazella, American Journal of the Medical Sciences, 2002,324(6):342-344).

Other candidates surfaced in clinical trials are Tenofovir-Alafenamide(TAF) and Hexadecyloxypropyl-Tenofovir (CMX-157), as illustrated inFIG. 1. TAF is an isopropylalaninyl phenyl ester that requires twodisparate enzymes for prodrug release: carboxyesterase and cathepsin A.The latter enzyme, cathepsin A, is a serine protease localized almostexclusively to lysosomal endosomes and ensures selective intracellulardelivery of TFV. TAF is currently approved in the clinic anddemonstrates little to no nephrotoxicity and more potent antiviralactivity than TDF at 1/10th the dose. Painter et al. reportedevaluations of CMX-157 as a potential treatment for HIV type 1 and HBVinfections (Antimicrob. Agents Chemother., 2007, 51, 3505-3509). CMX-157relies on the catalytic activity of an intracellularhydrolase-phospholipase C and/or sphingomylenase to liberate TFV withinthe cytosol. Available preliminary data indicates CMX-157 iswell-tolerated and achieves significant concentrations of TFVdpp up toone week after a single 400 mg dose, indicating the potential for aconvenient, once-a-week dosing regimen. However, CMX-157 has made littleprogression through the clinical trial pipeline since the completion ofPhase I in 2011.

Gosselin and collaborators previously examined dithioethanol (DTE)conjugates to mediate the delivery of adefovir, AZT, and 3′-deoxyuridine(ddU) to HIV-1 infected cell lines in vitro. U.S. Pat. No. 6,020,482reports phosphotriester type biologically active compounds and also seeU.S. Pat. Nos. 6,555,676, 7,902,202, and 8,871,785.

Conjugation of bis(DTE) to adefovir increases the HIV-1 activity of theparent nucleoside by ten-fold and confers exceptional stability(t_(1/2)>24 h) at pH 2, pH 7.4, culture medium, and human gastric juice.However, these conjugates rapidly degrade in human serum (t_(1/2)<5 min)which significantly limits their clinical utility. A proposed cleavagemechanism for these masking groups is illustrated in FIG. 2. Reductionof compound (i) releases β-mercaptoethanol and metastable intermediate(ii) that spontaneously collapses on the thioethanol linker to generatethiirane and the free nucleoside (iii) (when R═H). It was speculatedthat the β-mercaptoethyl linker may be a precursor for thiirane whichhas been implicated in the decomposition of S-acyl-2-thioethyl (SATE)and dithioethanol (DTE) prodrugs. Of note, SATE prodrugs also passthrough common intermediate (ii) following hydrolysis of an S-acylmoiety via non-specific carboxyesterases to liberate the targetnucleoside. The apparent instability of the disulfide linkage in serumhas stalled efforts to advance this technology forward and the mutagenicpotential of thiirane has precluded the clinical use of SATE andDTE-bound nucleosides. References cited herein are not an admission ofprior art.

Thus, there is a need to identify improvements and exploit alternativeprodrug strategies to enhance intracellular delivery ofpharmacologically active compounds, such as useful nucleosides liketenofovir.

SUMMARY

This disclosure relates to lipid disulfide prodrugs and in particular tolipid disulfide phosphodiester nucleosides and derivatives thereof,pharmaceutical compositions, and uses related thereto. According to oneembodiment of the disclosure there is provided a compound of Formula I,

or pharmaceutically acceptable salts or derivatives thereof, wherein, Wis a pharmacologically active compound, or linking group for connectingto a pharmacologically active compound; Z is selected from O, S, or Se;Y is selected from O, S, or NH; T is an aryl or alkyl linking group, R¹is a lipid; R² is R¹SST-, hydrogen, alkyl, aryl, phenyl, 4-fluorophenyl,4-chlorophenyl, 4-bromophenyl, naphthyl, or heterocyclyl, wherein R² isoptionally substituted with one or more, the same or different, R¹⁰;R¹⁰ is deuterium, alkyl, alkenyl, alkynyl, alkanoyl, halogen, nitro,cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkylsulfonyl, arylsulfonyl, benzyl, benzoyl, carbocyclyl, aryl, orheterocyclyl, wherein R¹⁰ is optionally substituted with one or more,the same or different, R¹¹; andR¹¹ is deuterium, halogen, nitro, cyano, hydroxy, trifluoromethoxy,trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl,methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethyl sulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,benzyl, benzoyl, carbocyclyl, aryl, or heterocyclyl.

Further features of this embodiment provide for W to be a nucleoside ornucleobase or a linking group for connecting to a nucleoside ornucleobase; T to be a C₂ to C₆ alkyl or an aryl, and T may be optionallysubstituted with one or more, the same or different R¹⁰; Z and Y areeach O; R¹ is a C₆ to C₂₀ lipid; R² is R¹—SST-, hydrogen, alkyl, aryl orphenyl. Yet further features of this embodiment provide for R¹ to be aC₁₀-C₁₈ lipid, preferably a C₁₆ to C₁₈ lipid, and more preferably a C₁₆lipid and for R² to be H, methyl, or alkyl.

In another embodiment of the disclosure, there is provided a compound ofFormula I′

or salts or derivatives thereof wherein, n is 2, 3, 4, 5 or 6; W is anucleoside or linking group for connecting to a nucleoside ornucleobase; Z is selected from O, S, or Se; Y is selected from O, S, orNH; R¹ is a lipid; R² is R¹SS(CH₂)_(n)—, hydrogen, alkyl, aryl, phenyl,4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, orheterocyclyl, wherein R² is optionally substituted with one or more, thesame or different, R¹⁰, as described above and which is optionallysubstituted with one or more, the same or different, R¹¹, as describedabove.

In another embodiment of the disclosure there is provided a compound ofFormula I″,

or salts or derivatives thereof wherein, T is —C₆H₄—, —(CH₂)_(n)—,—CH₂—C₆H₄—, a linking group providing an atomic chain of two, three orfour atoms, —R_(m)—, wherein m is 1, 2, 3, or 4, or a bridging alkyl,alkenyl, optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, substituted heterocyclyl; Uis N or CH; V is N or CR⁷; W is O or S; Z is selected from O, S, or Se;Y is selected from O, S, or NH; R¹ is a lipid; R² is R¹SST- or H; R⁴ ishydrogen, alkyl, or halogen, wherein R⁴ is optionally substituted withone or more, the same or different, R¹⁰; R⁶ is hydrogen, alkyl, amino,or halogen, wherein R⁶ is optionally substituted with one or more, thesame or different, R¹⁰; R⁷ is hydrogen, alkyl, or halogen, wherein R⁷ isoptionally substituted with one or more, the same or different, R¹⁰; R⁸is hydrogen, alkyl, amino, or halogen, wherein R⁸ is optionallysubstituted with one or more, the same or different, R¹⁰; R⁹ ishydrogen, alkyl, cyclopropyl, or carbocyclyl, wherein R⁹ is optionallysubstituted with one or more, the same or different, R¹⁰; R¹⁰ is asdecribed above and is optionally substituted with one or more, the sameor different, R¹¹, as described above.

In a preferred embodiment of the disclosure the compound of Formula Imay be selected from the following compounds as free acids, salts orderivatives thereof:

In a more preferred embodiment of the disclosure the compound of FormulaI may be selected from the following compounds as free acids, salts, orderivatives thereof:

In a further embodiment of the disclosure there is provided a method oftreating a viral infection comprising administering in effective amountof a compound as described above to a subject in need thereof.

In certain embodiments, the disclosure contemplates derivatives ofcompounds disclosed herein such as those containing one or more, thesame or different, substituents.

In certain embodiments, the disclosure contemplates pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and acompound disclosed herein. In certain embodiments, the pharmaceuticalcomposition is in the form of a tablet, capsule, pill, gel, granules,aerosol, or aqueous buffer, such as a saline or phosphate buffer, or ananoparticle formulation, emulsion, liposome, etc.

In certain embodiments, the disclosure relates to methods of preparingcompounds disclosed herein comprising mixing one or more startingmaterials with reagents under conditions such that the products areformed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates known prodrug conjugates of tenofovir:Tenofovir-Alafenamide (TAF) and Hexadecyloxypropyl-Tenofovir (CMX-157)in the clinic and clinical trials pipeline;

FIG. 2 illustrates a proposed mechanism for prodrug cleavage ofdithioethanol (DTE) conjugates;

FIG. 3 illustrates some embodiments of this disclosure;

FIG. 4A illustrates a first synthetic strategy for some embodiments ofthis disclosure;

FIG. 4B illustrates a second synthetic strategy for some embodiments ofthis disclosure;

FIG. 5 illustrates further embodiments of this disclosure; and

FIG. 6 illustrates methods for preparing further embodiments of thisdisclosure.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

To the extent that any chemical formulas reported herein contain one ormore chiral centers, the formulas are intended to encompass all stablestereoisomers, enantiomers, and diastereomers. It is also understoodthat formula encompass all tautomeric forms.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a”, “an”, and “the” include plural referentsunless the context clearly dictates otherwise.

“Subject” refers any animal, preferably a human patient, livestock,mouse model or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, a “lipid” group refers to a hydrophobic group that isnaturally or non-naturally occurring that is an alkyl chain of greaterthan six carbons or highly insoluble in water. As used herein a lipidgroup is considered highly insoluble in water when the point ofconnection on the lipid group is replaced with a hydrogen and theresulting compound has a solubility of less than 0.63×10⁻⁴% w/w (at 25°C.) in water, which is the percent solubility of octane in water byweight (see Solvent Recovery Handbook, 2nd Ed, Smallwood, 2002 byBlackwell Science, page 195). Examples of naturally occurring lipidsinclude, but are not limited to, saturated or unsaturated hydrocarbonchains found in fatty acids, glycerolipids, cholesterol, steroids,polyketides, and derivatives. Non-limiting examples of non-naturallyoccurring lipids include, but are not limited to, derivatives ofnaturally occurring lipids, acrylic polymers, aromatic, and alkylatedcompounds and derivatives thereof as well as those described herein.

A “linking group” refers to any variety of molecular arrangements thatcan be used to bridge to molecular moieties together. An example formulamay be —R_(m)— wherein R is selected individually and independently ateach occurrence as: —CR_(m)R_(m)—, —CHR_(m)—, —CH—, —C—, —CH₂—,—C(OH)R_(m), —C(OH)(OH)—, —C(OH)H, —C(Hal)R_(m)—, —C(Hal)(Hal)-,—C(Hal)H—, —C(N₃)R_(m)—, —C(CN)R_(m)—, —C(CN)(CN)—, —C(CN)H—,—C(N₃)(N₃)—, —C(N₃)H—, —O—, —S—, —N—, —NH—, —NR_(m)—, —(C═O)—, —(C═NH)—,—(C═S)—, —(C═CH₂)—, which may contain single, double, or triple bondsindividually and independently between the R groups. If an R is branchedwith an R_(m) it may be terminated with a group such as —CH₃, —H,—CH═CH₂, —CCH, —OH, —SH, —NH₂, —N₃, —CN, or —Hal, NO₂, SO₂R or twobranched Rs may form a cyclic structure. It is contemplated that incertain instances, the total Rs or “m” may be less than 100 or 50 or 25or 10. Examples of linking groups include, but are not limited to,bridging alkyl groups, alkoxyalkyl groups and aminoalkyl groups.

As used herein, “alkyl” means a noncyclic straight chain or branched,unsaturated or saturated hydrocarbon such as those containing from 1 to20 carbon atoms. A “higher alkyl” refers to unsaturated or saturatedhydrocarbon having 6 or more carbon atoms. A “C₈-C₁₈” refers to an alkylcontaining 8 to 18 carbon atoms. Likewise, a “C₆-C₂₂” refers to an alkylcontaining 6 to 22 carbon atoms. Representative saturated straight chainalkyls include but are methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, n-septyl, n-octyl, n-nonyl, hexadecyl, dodecyl, tetradecyl,izosonyl, octadecyl, and the like; while saturated branched alkylsinclude, but are not limited to, isopropyl, sec-butyl, isobutyl,tert-butyl, isopentyl, and the like. Unsaturated alkyls contain at leastone double or triple bond between adjacent carbon atoms (referred to asan “alkenyl” or “alkynyl”, respectively). Representative straight chainand branched alkenyls include, but are not limited to, ethylenyl,propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike; while representative straight chain and branched alkynyls include,but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as“carbocycles” or “carbocyclyl” groups. Representative saturatedcarbocycles include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like; while unsaturated carbocyclesinclude, but are not limited to, cyclopentenyl and cyclohexenyl, and thelike. Carbocyclyls include, but are not limited to, cycloalkyls andcycloalkenyls.

“Heterocarbocycles” or “heterocarbocyclyl” groups are carbocycles whichcontain from 1 to 4 heteroatoms independently selected from nitrogen,phosphorous, oxygen and sulphur which may be saturated or unsaturated(but not aromatic), monocyclic or polycyclic, and wherein the nitrogenand sulphur heteroatoms may be optionally oxidized (e.g. —S(O)—, —SO₂—,—N(O)—), and the nitrogen heteroatom may be optionally quaternized.Heterocarbocycles include, but are not limited to, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

The term “aryl” refers to aromatic homocyclic (i.e., hydrocarbon) mono-,bi- or tricyclic ring-containing groups preferably having 6 to 12 atomssuch as phenyl, naphthyl and biphenyl.

As used herein, “heteroaryl” or “heteroaromatic” refers an aromaticheterocarbocycle having 1 to 4 heteroatoms selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, including bothmono- and polycyclic ring systems. Polycyclic ring systems may, but arenot required to, contain one or more non-aromatic rings, as long as oneof the rings is aromatic. Representative heteroaryls are furyl,benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It iscontemplated that the use of the term “heteroaryl” includes N-alkylatedderivatives such as a 1-methylimidazol-5-yl substituent.

As used herein, “heterocycle” or “heterocyclyl” refers to mono- andpolycyclic ring systems having 1 to 4 heteroatoms selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom. Themono- and polycyclic ring systems may be aromatic, non-aromatic ormixtures of aromatic and non-aromatic rings. Heterocycle includesheterocarbocycles, heteroaryls, and the like.

“Alkoxy” refers to an alkyl group as defined above with the indicatednumber of carbon atoms attached through an oxygen bridge. Examples ofalkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, and s-pentoxy.Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, propoxy,n-butoxy, s-butoxy, t-butoxy.

“Alkoxyalkyl” refers an alkyl group as defined above with the indicatednumber of carbon atoms attached through an alkyl bridge (i.e.,—CH₂—O—CH₂CH₃).

“Alkylamino” refers an alkyl group as defined above with the indicatednumber of carbon atoms attached through an amino bridge. An example ofan alkylamino is methylamino, (i.e., —NH—CH₃).

“Alkylthio” refers to an alkyl group as defined above with the indicatednumber of carbon atoms attached through a sulfur bridge. An example ofan alkylthio is methylthio, (i.e., —S—CH₃).

“Alkanoyl” refers to an alkyl as defined above with the indicated numberof carbon atoms attached through a carbonyl bride (i.e., —(C═O)alkyl).

The terms “cycloalkyl” and “cycloalkenyl” refer to mono-, bi-, or trihomocyclic ring groups of 3 to 15 carbon atoms which are, respectively,fully saturated and partially unsaturated.

The terms “halogen” or “Hal” refer to fluorine, chlorine, bromine, andiodine.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents”. The molecule may be multiplysubstituted. In the case of an oxo substituent (═O), two hydrogen atomsare replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb,—NRaC(═O)NRaNRb, —NRaC(═O)ORb, —NRaSO₂Rb, —C(═O)Ra, —C(═O)ORa,—C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)₂Ra, —OS(═O)₂Ra and—S(═O)₂ORa. Ra and Rb in this context may be the same or different andindependently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino,alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl.

The term “optionally substituted”, as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom to be unsubstituted.

The term “nucleoside” refers to a non-aromatic five membered ringsubstituted, e.g., tetrahydrofuran-2-yl substituted in the 5 position,with a nucleobase or heterocyclic derivative. The five membered ringand/or nucleobase may be further substituted or derivatized. Examples ofnucleosides with modified adenosine or guanosine include, but are notlimited to, hypoxanthine, xanthine, 7-methylguanine. Examples ofnucleosides with modified cytidine, thymidine, or uridine include, butare not limited to, 5,6-dihydrouracil, 5-methylcytosine,5-hydroxymethylcytosine.

A “nucleobase” refers to any variety of nitrogen containing monocyclicor bicyclic heterocycles. Nucleobases typically have at least oneoptionally substituted amino group connected to the ring(s), or acarbonyl/hydroxyl group within the ring(s), or an optionally substitutedamide connected to the ring(s). It typically has two to four nitrogenatoms in the ring(s). Non-limiting examples of a nucleobase includeadenine, guanine, cytosine, uracil, thymine, inosine, and heterocyclesof the following structures:

With regard to the nucleobases, it is contemplated that the termencompasses isobases. Contemplated isobases include2′-deoxy-5-methylisocytidine (iC) and 2′-deoxy-isoguanosine (iG) (seeU.S. Pat. Nos. 6,001,983; 6,037,120; 6,617,106; and 6,977,161).

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedanalogue. The derivative may be structurally similar because it islacking one or more atoms, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, adding a hydroxylgroup, replacing an oxygen atom with a sulfur atom, or replacing anamino group with a hydroxyl group, oxidizing a hydroxyl group to acarbonyl group, reducing a carbonyl group to a hydroxyl group, andreducing a carbon-to-carbon double bond to an alkyl group or oxidizing acarbon-to-carbon single bond to a double bond. A derivative optionallyhas one or more, the same or different, substitutions. Derivatives maybe prepared by any variety of synthetic methods or appropriateadaptations presented in synthetic or organic chemistry text books, suchas those provided in “March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure”, Wiley, 6th Edition (2007) Michael B. Smithor “Domino Reactions in Organic Synthesis”, Wiley (2006) Lutz F. Tietze,hereby incorporated by reference.

Lipid Disulfide Nucleotide Derivative

Disulfide-linked lipid conjugates were designed to test for penetrationof the plasma membrane and release of the nucleoside within the targetcell.

Glutathione, often referred to as GSH, is a tripeptide typically inmillimolar concentrations (2-10 mM) within the cytosol that participatesin a multitude of biological functions to promote detoxification ofxenobiotics, mediates immunoregulation, and curbs oxidative stress,amongst others. GSH is seldom found in the extracellular space and thuscreates a gradient that fosters intracellular reduction of disulfidesand simultaneously promotes an oxidative environment to support S—Sformation in the surrounding media. Reduction-sensitive lipid conjugatesof tenofovir were developed (as shown in FIG. 3) that exhibitsub-nanomolar activity towards HIV-1 and are stable in human plasma formore than 24 h with a therapeutic index approaching 30,000.

In certain embodiments, a lipid disulfide nucleotide prodrug is acompound having Formula A,

or pharmaceutically acceptable salts or derivatives thereof, wherein, Tis a linking group;

W is a nucleoside or linking group for connecting to a nucleoside ornucleobase or, alkyl, alkenyl, alkynyl, alkanoyl, halogen, nitro, cyano,hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkylsulfonyl, arylsulfonyl, benzyl, benzoyl, carbocyclyl, aryl, orheterocyclyl, wherein W is optionally substituted with one or more, thesame or different, R¹⁰; X and Y are individually and independentlyselected from O, S, NH or Se; Z is S, O or Se; R¹ is a lipid; R² isR¹—SST-, hydrogen, alkyl, aryl, phenyl, 4-fluorophenyl, 4-chlorophenyl,4-bromophenyl, naphthyl, or heterocyclyl, wherein R² is optionallysubstituted with one or more, the same or different, R¹⁰, as describedabove, and wherein R¹⁰ is optionally substituted with one or more, thesame or different, R¹¹, as described above.

In certain embodiments, T is -Q-CH₂— and Q is —C₆H₄—, —(CH₂)_(n)—,—CH₂—C₆H₄—, a linking group providing an atomic chain of two, three orfour atoms, —R_(m)—, wherein m is 1, 2, 3, or 4, or a bridging alkyl,alkenyl, carbocyclyl, optionally substituted carbocyclyl,heterocarbocyclyl, optionally substituted heterocarbocyclyl, aryl,optionally substituted aryl, heterocyclyl, or optionally substitutedheterocyclyl.

In certain embodiments, a lipid disulfide derivative is a compoundhaving the following Formula B,

or salts or derivatives thereof wherein, Q is —C₆H₄—, —(CH₂)_(n)—,—CH₂—C₆H₄—, a linking group providing an atomic chain of two, three orfour atoms, —R_(m)—, wherein m is 1, 2, 3, or 4, or a bridging alkyl,alkenyl, carbocyclyl, optinally substituted carbocyclyl,heterocarbocyclyl, optionally substituted heterocarbocyclyl, aryl,optionally substituted aryl, heterocyclyl, or optionally substitutedheterocyclyl; n is 0, 1, 2, 3, 4, or 5; W is a nucleoside or linkinggroup for connecting to a nucleoside or nucleobase or, alkyl, alkenyl,alkynyl, alkanoyl, halogen, nitro, cyano, hydroxy, amino, amido,mercapto, formyl, carboxy, carbamoyl, azido, alkoxy, alkylthio,alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,benzyl, benzoyl, carbocyclyl, aryl, or heterocyclyl, wherein W isoptionally substituted with one or more, the same or different, R¹⁰; Xand Y are individually and independently selected from O, S, NH or Se; Zis S, O or Se; R¹ is a lipid; R² is R¹SSQCH₂—, hydrogen, alkyl, aryl,phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, orheterocyclyl, wherein R² is optionally substituted with one or more, thesame or different, R¹⁰, as described above, and wherein R¹⁰ isoptionally substituted with one or more, the same or different, R¹¹, asdescribed above.

In certain embodiments, a lipid disulfide derivative is a compoundhaving the following Formula C,

or salts or derivatives thereof wherein, n is 1, 2, 3, 4, or 5; W is anucleoside or linking group for connecting to a nucleoside or nucleobaseor, alkyl, alkenyl, alkynyl, alkanoyl, halogen, nitro, cyano, hydroxy,amino, amido, mercapto, formyl, carboxy, carbamoyl, azido, alkoxy,alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, benzyl, benzoyl, carbocyclyl, aryl, or heterocyclyl, wherein Wis optionally substituted with one or more, the same or different, R¹⁰;X and Y are individually and independently selected from O, S, NH or Se;Z is S, O or Se;

R¹ is a lipid; R² is R¹SS(CH₂)_(n)CH₂—, hydrogen, alkyl, aryl, phenyl,4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, orheterocyclyl, wherein R² is optionally substituted with one or more, thesame or different, R¹⁰, as described above, and wherein R¹⁰ isoptionally substituted with one or more, the same or different, R¹¹, asdescribed above.

In certain embodiments, R² is H, alkyl, or —CH₂(CH₂)_(n)SSR¹, morepreferably, R² is H, methyl, or alkyl.

In certain embodiments, a lipid disulfide derivative is a compoundhaving the following Formula D,

or salts or derivatives thereof wherein, T is a linking group or -QCH₂—;Q is —C₆H₄—, —(CH₂)_(n)—, —CH₂—C₆H₄—, a linking group providing anatomic chain of two, three or four atoms, —R_(m)—, wherein m is 1, 2, 3,or 4, or a bridging alkyl, alkenyl, carbocyclyl, optionally substitutedcarbocyclyl, heterocarbocyclyl, optionally substitutedheterocarbocyclyl, aryl, optionally substituted aryl, heterocyclyl, oroptionally substituted heterocyclyl; n is 1, 2, 3, 4, or 5; W is —CH₂O—,—CH₂S—, O, S, CH₂NH or NH;

X is O, S, or NH; Y is O, S, or NH; Z is O, S or Se;

-   -   R¹ is a lipid, alkyl, (C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher        alkyl, alkenyl, (C₈-C₁₈)alkenyl, (C₆-C₂₂)alkenyl, higher        alkenyl, alkynyl, (C₈-C₁₈)alkynyl, (C₆-C₂₂)alkynyl, or higher        alkynyl; or R¹ is aryl substituted with alkyl, (C₈-C₁₈)alkyl,        (C₆-C₂₂)alkyl, higher alkyl, alkenyl, (C₈-C₁₈)alkenyl,        (C₆-C₂₂)alkenyl, higher alkenyl, alkynyl, (C₈-C₁₈)alkynyl,        (C₆-C₂₂)alkynyl, or higher alkynyl wherein R¹ is optionally        substituted with one or more, the same or different, R¹⁰; R² is        R¹SST-, hydrogen, methyl, alkyl, aryl, phenyl, 4-fluorophenyl,        4-chlorophenyl, 4-bromophenyl, naphthyl, or heterocyclyl,        wherein R² is optionally substituted with one or more, the same        or different, R¹⁰; R³ is nucleotide, alkyl, alkoxyalkyl,        carbocyclyl, or heterocarbocyclyl substituted with a nucleobase        or a heterocyclyl, wherein R³ is optionally substituted with one        or more, the same or different, R¹⁰; or R³ is        tetrahydrofuran-2-yl or cyclopentenyl, substituted with a        nucleobase or a heterocyclyl, wherein R³ is optionally further        substituted with one or more, the same or different, R¹⁰,        wherein the optional further substitution is on the nucleotide,        alkyl, alkoxyalkyl, nucleobase, carbocyclyl, cyclopentenyl,        heterocarbocyclyl, tetrahydrofuran-2-yl, or heterocyclyl; R⁴ is        hydrogen, alkyl, halogen, or hydroxymethyl, wherein R⁴ is        optionally substituted with one or more, the same or different,        R¹⁰; or R² and R⁴ and attached atoms come together to form a six        membered heterocyclyl; or R³ and R⁴ and attached atoms come        together to form a five membered carbocyclic or        heterocarbocyclic ring substituted with a nucleobase or a        heterocyclyl which is optionally further substituted with one or        more, the same or different, R¹⁰, wherein the optional further        substitution is on the five membered carbocyclic or        heterocarbocyclic ring, nucleobase, or heterocyclyl; R¹⁰ is as        described above, and may be optionally substituted with one or        more, the same or different, R¹¹, as described above.

In certain embodiments, R²X is HO—, R¹SSTO—, methylO—, or alkylO—.

In certain embodiments, X and Y are O.

In certain embodiments, X and Y and Z are O.

In certain embodiments, X is NH and Y and Z are O.

In certain embodiments, W is —O—.

In certain embodiments, W is —O—, and R²X is HO—, R¹SSTO—, methyl-, oralkylO—.

In certain embodiments, W is —O—, and X and Y are O.

In certain embodiments, W is —O—, and X and Y and Z are O.

In certain embodiments, W is —O—, and X is NH and Y and Z are O, and R²is H, R¹SSTO-, methyl-, or alkyl-.

In certain embodiments, W is O, and R²X is HO—, R¹SSTO—, methylO—, oralkylO—.

In certain embodiments, W is O, and X and Y are O, and R²X is HO—,R¹SSTO—, methylO—, or alkylO—.

In certain embodiments, W is O, and X and Y and Z are O, and R²X is HO—,R¹SSTO—, methylO—, or alkylO—.

In certain embodiments, W is O, and X is NH and Y and Z are O, and R² isH, R¹SSTO—, methyl-, or alkyl-.

In certain embodiments, a lipid disulfide derivative is a compoundhaving the following Formula E,

or salts or derivatives thereof wherein, Q is —C₆H₄—, —(CH₂)_(n)—,—CH₂—C₆H₄—, a linking group providing an atomic chain of two, three orfour atoms, —R_(m)—, wherein m is 1, 2, 3, 4 or 5, or a bridging alkyl,alkenyl, carbocyclyl, optionally substituted carbocyclyl,heterocarbocyclyl, optionally substituted heterocarbocyclyl, aryl,optionally substituted aryl, heterocyclyl, or optionally substitutedheterocyclyl; n is 1, 2, 3, 4, or 5; W is —CH₂O—, —CH₂S—, O, or S; X isO, S, or NH; Y is O, S, or NH; Z is O or S; R¹ is a lipid, alkyl,(C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl, alkenyl, (C₈-C₁₈)alkenyl,(C₆-C₂₂)alkenyl, higher alkenyl, alkynyl, (C₈-C₁₈)alkynyl,(C₆-C₂₂)alkynyl, or higher alkynyl; or R¹ is aryl substituted withalkyl, (C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl, alkenyl,(C₈-C₁₈)alkenyl, (C₆-C₂₂)alkenyl, higher alkenyl, alkynyl,(C₈-C₁₈)alkynyl, (C₆-C₂₂)alkynyl, or higher alkynyl wherein R¹ isoptionally substituted with one or more, the same or different, R¹⁰; R²is R¹SSQCH₂—, hydrogen, methyl, alkyl, aryl, phenyl, 4-fluorophenyl,4-chlorophenyl, 4-bromophenyl, naphthyl, or heterocyclyl, wherein R² isoptionally substituted with one or more, the same or different, R¹⁰; R³is nucleotide, alkyl, alkoxyalkyl, carbocyclyl, or heterocarbocyclylsubstituted with a nucleobase or a heterocyclyl, wherein R³ isoptionally substituted with one or more, the same or different, R¹⁰; orR³ is tetrahydrofuran-2-yl or cyclopentenyl, substituted with anucleobase or a heterocyclyl, wherein R³ is optionally furthersubstituted with one or more, the same or different, R¹⁰, wherein theoptional further substitution is on the nucleotide, alkyl, alkoxyalkyl,nucleobase, carbocyclyl, cyclopentenyl, heterocarbocyclyl,tetrahydrofuran-2-yl, or heterocyclyl; R⁴ is hydrogen, alkyl, halogen,or hydroxymethyl, wherein R⁴ is optionally substituted with one or more,the same or different, R¹⁰; or R² and R⁴ and attached atoms cometogether to form a six membered heterocyclyl; or R³ and R⁴ and attachedatoms come together to form a five membered carbocyclic orheterocarbocyclic ring substituted with a nucleobase or a heterocyclylwhich is optionally further substituted with one or more, the same ordifferent, R¹⁰, wherein the optional further substitution is on the fivemembered carbocyclic or heterocarbocyclic ring, nucleobase, orheterocyclyl; R¹⁰ is as described above, and may be optionallysubstituted with one or more, the same or different, R¹¹, as describedabove.

In certain embodiments, R²X is HO—, R¹SSQCH₂O—, methylO—, or alkylO—.

In certain embodiments, X and Y are O.

In certain embodiments, X and Y and Z are O.

In certain embodiments, X is NH and Y and Z are O.

In certain embodiments, W is —O—.

In certain embodiments, W is —O—, and R²X is HO—, R¹SSQCH₂O—, methyl-,or alkylO—.

In certain embodiments, W is —O—, and X and Y are O.

In certain embodiments, W is —O—, and X and Y and Z are O.

In certain embodiments, W is —O—, and X is NH and Y and Z are O, and R²is H, R¹SSQCH₂O—, methyl-, or alkyl-.

In certain embodiments, W is O, and R²X is HO—, R¹SSQCH₂O—, methylO—, oralkylO—.

In certain embodiments, W is O, and X and Y are O, and R²X is HO—,R¹SSQCH₂O—, methylO—, or alkylO—.

In certain embodiments, W is O, and X and Y and Z are O, and R²X is HO—,R¹SSQCH₂O—, methylO—, or alkylO—.

In certain embodiments, W is O, and X is NH and Y and Z are O, and R² isH, R¹SSOCH₂O—, methyl-, or alkyl-.

In certain embodiments, a lipid disulfide derivative is a compoundhaving the following Formula F,

or salts or derivatives thereof wherein, n is 1, 2, 3, 4, or 5; W is—CH₂O—, —CH₂S—, O, or S; X is O, S, or NH; Y is O, S, or NH; Z is O orS; R¹ is a lipid, alkyl, (C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl,alkenyl, (C₈-C₁₈)alkenyl, (C₆-C₂₂)alkenyl, higher alkenyl, alkynyl,(C₈-C₁₈)alkynyl, (C₆-C₂₂)alkynyl, or higher alkynyl; or R¹ is arylsubstituted with alkyl, (C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl,alkenyl, (C₈-C₁₈)alkenyl, (C₆-C₂₂)alkenyl, higher alkenyl, alkynyl,(C₈-C₁₈)alkynyl, (C₆-C₂₂)alkynyl, or higher alkynyl wherein R¹ isoptionally substituted with one or more, the same or different, R¹⁰; R²is R¹SS(CH₂)_(n)CH₂—, hydrogen, methyl, alkyl, aryl, phenyl,4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, orheterocyclyl, wherein R² is optionally substituted with one or more, thesame or different, R¹⁰; R³ is nucleotide, alkyl, alkoxyalkyl,carbocyclyl, or heterocarbocyclyl substituted with a nucleobase or aheterocyclyl, wherein R³ is optionally substituted with one or more, thesame or different, R¹⁰; or R³ is tetrahydrofuran-2-yl or cyclopentenyl,substituted with a nucleobase or a heterocyclyl, wherein R³ isoptionally further substituted with one or more, the same or different,R¹⁰, wherein the optional further substitution is on the nucleotide,alkyl, alkoxyalkyl, nucleobase, carbocyclyl, cyclopentenyl,heterocarbocyclyl, tetrahydrofuran-2-yl, or heterocyclyl; R⁴ ishydrogen, alkyl, halogen, or hydroxymethyl, wherein R⁴ is optionallysubstituted with one or more, the same or different, R¹⁰; or R² and R⁴and attached atoms come together to form a six membered heterocyclyl; orR³ and R⁴ and attached atoms come together to form a five memberedcarbocyclic or heterocarbocyclic ring substituted with a nucleobase or aheterocyclyl which is optionally further substituted with one or more,the same or different, R¹⁰, wherein the optional further substitution ison the five membered carbocyclic or heterocarbocyclic ring, nucleobase,or heterocyclyl; R¹⁰ is as described above, and wherein R¹⁰ isoptionally substituted with one or more, the same or different, R¹¹, asdescribed above.

In certain embodiments, R²X is HO—, R¹SS(CH₂)_(n)CH₂O—, methylO—, oralkylO—.

In certain embodiments, X and Y are O.

In certain embodiments, X and Y and Z are O.

In certain embodiments, X is NH and Y and Z are O.

In certain embodiments, W is —CH₂O—.

In certain embodiments, W is —CH₂O—, and R²X is HO—, R¹SS(CH₂)_(n)CH₂O—,methyl-, or alkylO—.

In certain embodiments, W is —CH₂O—, and X and Y are O.

In certain embodiments, W is —CH₂O—, and X and Y and Z are O.

In certain embodiments, W is —CH₂O—, and X is NH and Y and Z are O, andR² is H, R¹SS(CH₂)_(n)CH₂O—, methyl-, or alkyl-.

In certain embodiments, W is O.

In certain embodiments, W is O, and R²X is HO—, R¹SS(CH₂)_(n)CH₂O—,methylO—, or alkylO—.

In certain embodiments, W is O, and X and Y are O, and R²X is HO—,R¹SS(CH₂)_(n)CH₂O—, methylO—, or alkylO—.

In certain embodiments, W is O, and X and Y and Z are O, and R²X is HO—,R¹SS(CH₂)_(n)CH₂O—, methylO—, or alkylO—.

In certain embodiments, W is O, and X is NH and Y and Z are O, and R² isH, R¹SS(CH₂)_(n)CH₂O—, methyl-, or alkyl-.

In certain embodiments, a lipid disulfide derivative is a compoundhaving the following Formula,

or salts or derivatives thereof wherein, Q is —C₆H₄—, —(CH₂)_(n)—,—CH₂—C₆H₄—, a linking group providing an atomic chain of two, three orfour atoms, —R_(m)—, wherein m is 1, 2, 3, or 4, or a bridging alkyl,alkenyl, carbocyclyl, optionally substituted carbocyclyl,heterocarbocyclyl, optionally substituted heterocarbocyclyl, aryl,optionally substituted aryl, heterocyclyl, or optionally substitutedheterocyclyl; n is 1, 2, 3, 4, or 5; W is —CH₂O—, —CH₂S—, O, or S; X isO, S, or NH; Y is O, S, or NH; Z is O or S; R¹ is a lipid, alkyl,(C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl, alkenyl, (C₈-C₁₈)alkenyl,(C₆-C₂₂)alkenyl, higher alkenyl, alkynyl, (C₈-C₁₈)alkynyl,(C₆-C₂₂)alkynyl, or higher alkynyl; or R¹ is aryl substituted withalkyl, (C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl, alkenyl,(C₈-C₁₈)alkenyl, (C₆-C₂₂)alkenyl, higher alkenyl, alkynyl,(C₈-C₁₈)alkynyl, (C₆-C₂₂)alkynyl, or higher alkynyl wherein R¹ isoptionally substituted with one or more, the same or different, R¹⁰; R²is R¹SSQCH₂—, hydrogen, alkyl, aryl, phenyl, 4-fluorophenyl,4-chlorophenyl, 4-bromophenyl, naphthyl, or heterocyclyl, wherein R² isoptionally substituted with one or more, the same or different, R¹⁰; R⁵is a nucleobase or a heterocyclyl, wherein R⁵ is optionally substitutedwith one or more, the same or different, R¹⁰; R⁴ is hydrogen, alkyl,halogen, or hydroxymethyl, wherein R⁴ is optionally substituted with oneor more, the same or different, R¹⁰; R¹⁰ is as described above, andwherein R¹⁰ is optionally substituted with one or more, the same ordifferent, R¹¹, as described above.

In certain embodiments, a lipid dithiol phosphodiester nucleotidederivative is a compound having the following Formula,

or salts or derivatives thereof wherein, Q is —C₆H₄—, —(CH₂)_(n)—,—CH₂—C₆H₄—, a linking group providing an atomic chain of two, three orfour atoms, —R_(m)—, wherein m is 1, 2, 3, 4, or 5, or a bridging alkyl,alkenyl, carbocyclyl, substituted carbocyclyl, heterocarbocyclyl,substituted heterocarbocyclyl, aryl, substituted aryl, heterocyclyl, orsubstituted heterocyclyl; n is 1, 2, 3, 4, or 5; U is N or CH; V is N orCH; W is O or S; X is O, S, NH or Se; Y is O, S, NH or Se; Z is O, S orSe; R¹ is a lipid, alkyl, (C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl,alkenyl, (C₈-C₁₈)alkenyl, (C₆-C₂₂)alkenyl, higher alkenyl, alkynyl,(C₈-C₁₈)alkynyl, (C₆-C₂₂)alkynyl, or higher alkynyl; or R¹ is arylsubstituted with alkyl, (C₈-C₁₈)alkyl, (C₆-C₂₂)alkyl, higher alkyl,alkenyl, (C₈-C₁₈)alkenyl, (C₆-C₂₂)alkenyl, higher alkenyl, alkynyl,(C₈-C₁₈)alkynyl, (C₆-C₂₂)alkynyl, or higher alkynyl wherein R¹ isoptionally substituted with one or more, the same or different, R¹⁰; R²is R¹SSQCH₂—, hydrogen, alkyl, aryl, phenyl, 4-fluorophenyl,4-chlorophenyl, 4-bromophenyl, naphthyl, or heterocyclyl, wherein R² isoptionally substituted with one or more, the same or different, R¹⁰; R⁴is hydrogen, alkyl, halogen, or hydroxymethyl, wherein R⁴ is optionallysubstituted with one or more, the same or different, R¹⁰; R⁶ ishydrogen, alkyl, amino, or halogen, wherein R⁶ is optionally substitutedwith one or more, the same or different, R¹⁰; R⁷ is hydrogen, alkyl, orhalogen, wherein R⁷ is optionally substituted with one or more, the sameor different, R¹⁰; R⁸ is hydrogen, alkyl, amino, or halogen, wherein R⁸is optionally substituted with one or more, the same or different, R¹⁰;R⁹ is hydrogen, alkyl, cyclopropyl, or carbocyclyl, wherein R⁹ isoptionally substituted with one or more, the same or different, R¹⁰; R¹⁰is as described above, and wherein R¹⁰ is optionally substituted withone or more, the same or different, R¹¹, as described above.

Exemplary compounds include:

Methods of Preparation

Preparation of compounds disclosed herein may be prepared usingprocedures as outlined in the Figures. In certain embodiments, thedisclosure relates to methods of preparing compounds of Formula Acomprising mixing the following compounds,

under conditions such that a compound of Formula A is formed,

wherein the substitutents T, W, X, Y, Z, R¹ and R² are reported herein.

In certain embodiments, the disclosure relates to methods of preparingcompounds of formula WC comprising mixing the following compounds,

under conditions such that a compound of formula WC is formed,

wherein the substituents, e.g., Q W, X, Z, R¹ and R² are reportedherein.

In certain embodiments, the disclosure relates to methods of preparingcompounds of formula WE comprising mixing the following compounds,

under conditions such that a compound of formula WE is formed,

wherein the substituents, e.g., n, W, X, Z, R¹ and R² are reportedherein.

Methods of Use

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection or cancer comprising administering ineffective amount of a compound disclosed herein to a subject in needthereof. In some embodiments, the subject is at risk of, exhibitingsymptoms of, suffering from, or diagnosed with a viral infection.

In some embodiments, the subject is at risk of, exhibiting symptoms of,or diagnosed with influenza A virus including subtype H1N1, influenza Bvirus, influenza C virus, rotavirus A, rotavirus B, rotavirus C,rotavirus D, rotavirus E, SARS coronavirus, Respiratory syncytial virus(RSV), human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV)Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, parvovirus B19,molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cellpolyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocyticchoriomeningitis virus (LCMV), yellow fever virus, measles virus, mumpsvirus, rinderpest virus, California encephalitis virus, hantavirus,rabies virus, ebola virus, marburg virus, herpes simplex virus-1(HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV),Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropicvirus, roseolovirus, Kaposi's sarcoma-associated herpesvirus, hepatitisA (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV),hepatitis E (HEV), human immunodeficiency virus (HIV), The HumanT-lymphotropic virus Type I (HTLV-1), Friend spleen focus-forming virus(SFFV) or Xenotropic MuLV-Related Virus (XMRV).

In certain embodiments, the viral infection is an alphavirus, flavivirusor coronaviruses orthomyxoviridae or paramyxoviridae, Powassan virus orfiloviridae. In certain embodiments, the viral infection is selectedfrom MERS coronavirus, Eastern equine encephalitis virus, Western equineencephalitis virus, Venezuelan equine encephalitis virus, Ross Rivervirus, and Chikungunya virus.

In certain embodiments, methods disclosed herein are contemplated to beadministered in combination with other the antiviral agent(s) such asabacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen,arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, complera,darunavir, delavirdine, didanosine, docosanol, dolutegravir, edoxudine,efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir,fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir,ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine,interferon type III, interferon type II, interferon type I, lamivudine,lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir,nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir,peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin,rimantadine, ritonavir, pyramidine, saquinavir, stavudine, stribild,tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, or zidovudine, and combinations thereof.

In certain embodiments, the disclosure contemplates the treatment orprevention of a viral infection using compounds disclosed herein,wherein viral infection is human immunodeficiency virus or hepatitis Bvirus.

In certain embodiments, the disclosure relates to methods of treatingcancer comprising administering in effective amount of a compounddisclosed herein to a subject in need thereof.

“Cancer” refers any of various cellular diseases with malignantneoplasms characterized by the proliferation of cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Within the contextof certain embodiments, whether “cancer is reduced” may be identified bya variety of diagnostic manners known to one skill in the art including,but not limited to, observation the reduction in size or number of tumormasses or if an increase of apoptosis of cancer cells observed, e.g., ifmore than a 5% increase in apoptosis of cancer cells is observed for asample compound compared to a control without the compound. It may alsobe identified by a change in relevant biomarker or gene expressionprofile, such as PSA for prostate cancer, HER2 for breast cancer, orothers.

The cancer to be treated in the context of the present disclosure may beany type of cancer or tumor. These tumors or cancer include, and are notlimited to, tumors of the hematopoietic and lymphoid tissues orhematopoietic and lymphoid malignancies, tumors that affect the blood,bone marrow, lymph, and lymphatic system. Hematological malignancies mayderive from either of the two major blood cell lineages: myeloid andlymphoid cell lines. The myeloid cell line normally producesgranulocytes, erythrocytes, thrombocytes, macrophages and mast cells;the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas,lymphocytic leukemias, and myeloma are from the lymphoid line, whileacute and chronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin.

Also contemplated are malignancies located in the colon, abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvis, skin, soft tissue, spleen, thorax and genito-urinaryapparatus and, more particularly, childhood acute lymphoblasticleukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia,acute myeloid leukemia, adrenocortical carcinoma, adult (primary)hepatocellular cancer, adult (primary) liver cancer, adult acutelymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin'sdisease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adultnon-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissuesarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, analcancer, astrocytoma, cancer of the biliary tract, cancer of the bladder,bone cancer, brain stem glioma, brain tumors, breast cancer, cancer ofthe renal pelvis and ureter, primary central nervous system lymphoma,central nervous system lymphoma, cerebellar astrocytoma, brainastrocytoma, cancer of the cervix, childhood (primary) hepatocellularcancer, childhood (primary) liver cancer, childhood acute lymphoblasticleukemia, childhood acute myeloid leukemia, childhood brain stem glioma,childhood cerebellar astrocytoma, childhood brain astrocytoma, childhoodextracranial germ cell tumors, childhood Hodgkin's disease, childhoodHodgkin's lymphoma, childhood visual pathway and hypothalamic glioma,childhood lymphoblastic leukemia, childhood medulloblastoma, childhoodnon-Hodgkin's lymphoma, childhood supratentorial primitiveneuroectodermal and pineal tumors, childhood primary liver cancer,childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhoodvisual pathway and hypothalamic glioma, chronic lymphocytic leukemia,chronic myeloid leukemia, cancer of the colon, cutaneous T-celllymphoma, endocrine pancreatic islet cells carcinoma, endometrialcancer, ependymoma, epithelial cancer, cancer of the oesophagus, Ewing'ssarcoma and related tumors, cancer of the exocrine pancreas,extracranial germ cell tumor, extragonadal germ cell tumor, extrahepaticbiliary tract cancer, cancer of the eye, breast cancer in women,Gaucher's disease, cancer of the gallbladder, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal tumors, germ celltumors, gestational trophoblastic tumor, tricoleukemia, head and neckcancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma,hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers,intraocular melanoma, islet cell carcinoma, islet cell pancreaticcancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancerof the lip and mouth, cancer of the liver, cancer of the lung,lymphoproliferative disorders, macroglobulinemia, breast cancer in men,malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma,mesothelioma, occult primary metastatic squamous neck cancer, primarymetastatic squamous neck cancer, metastatic squamous neck cancer,multiple myeloma, multiple myeloma/plasmatic cell neoplasia,myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia,myeloproliferative disorders, paranasal sinus and nasal cavity cancer,nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma duringpregnancy, non-melanoma skin cancer, non-small cell lung cancer,metastatic squamous neck cancer with occult primary, buccopharyngealcancer, malignant fibrous histiocytoma, malignant fibrousosteosarcoma/histiocytoma of the bone, epithelial ovarian cancer,ovarian germ cell tumor, ovarian low malignant potential tumor,pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancerof the penis, phaeochromocytoma, hypophysis tumor, neoplasia ofplasmatic cells/multiple myeloma, primary central nervous systemlymphoma, primary liver cancer, prostate cancer, rectal cancer, renalcell cancer, cancer of the renal pelvis and ureter, retinoblastoma,rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas,skin cancer, small cell lung cancer, small intestine cancer, soft tissuesarcoma, squamous neck cancer, stomach cancer, pineal and supratentorialprimitive neuroectodermal tumors, T-cell lymphoma, testicular cancer,thymoma, thyroid cancer, transitional cell cancer of the renal pelvisand ureter, transitional renal pelvis and ureter cancer, trophoblastictumors, cell cancer of the renal pelvis and ureter, cancer of theurethra, cancer of the uterus, uterine sarcoma, vaginal cancer, opticpathway and hypothalamic glioma, cancer of the vulva, Waldenstrom'smacroglobulinemia, Wilms' tumor and any other hyperproliferativedisease, as well as neoplasia, located in the system of a previouslymentioned organ.

In certain embodiments, this disclosure contemplates administeringcompounds reported herein in combined with another chemotherapy regimenor anticancer agent.

A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent” or thelike, refer to molecules that are recognized to aid in the treatment ofa cancer. Contemplated examples include combinations such as a compounddisclosed herein and cyclophosphamide, methotrexate, 5-fluorouracil(CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine,procarbazine, prednisolone (MOPP); sdriamycin, bleomycin, vinblastine,dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine,prednisolone (CHOP); rituximab, cyclophosphamide, doxorubicin,vincristine, prednisolone (RCHOP); bleomycin, etoposide, cisplatin(BEP); epirubicin, cisplatin, 5-fluorouracil (ECF); or epirubicin,cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin,cisplatin (MVAC).

In certain embodiments, the anti-cancer agent selected from abemaciclib,abiraterone acetate, methotrexate, paclitaxel, adriamycin,acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine,aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin,alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan,brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole,apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine,arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab,axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat,bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene,bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib,brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine,fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin,cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine,cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib,ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine,decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukindiftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel,doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab,oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin,vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene,panobinostat, fulvestrant, letrozole, filgrastim, fludarabine,flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine,gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol,trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib,ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec,ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel,palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide,lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide,lomustine, trifluridine, olaparib, vincristine, procarbazine,mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexonebromide, midostaurin, mitomycin C, mitoxantrone, plerixafor,vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib,niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine,pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib,interferon alfa-2b, pertuzumab, pomalidomide, mercaptopurine,regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib,thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin,valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronicacid, or combinations thereof.

In certain embodiments, the chemotherapy agent is an anti-PD-1,anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4(e.g., ipilimumab, tremelimumab) and anti-PD1 (e.g., nivolumab,pembrolizumab, atezolizumab, avelumab, durvalumab). In certainembodiments, the method of administration is in a subject with alymphodepleted environment. In certain embodiments, lymphodepletingagents (e.g., cyclophosphamide and fludarabine)

Formulations

Pharmaceutical compositions disclosed herein may be in the form ofpharmaceutically acceptable salts, as generally described below. Somepreferred, but non-limiting examples of suitable pharmaceuticallyacceptable organic and/or inorganic acids are hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citricacid, as well as other pharmaceutically acceptable acids known per se(for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well asa basic group, the compounds of the disclosure may also form internalsalts, and such compounds are within the scope of the disclosure. When acompound contains a hydrogen-donating heteroatom (e.g. NH), salts arecontemplated to covers isomers formed by transfer of said hydrogen atomto a basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds include the acidaddition and base salts thereof. Suitable acid addition salts are formedfrom acids which form non-toxic salts. Examples include the acetate,adipate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate,esylate, formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate,saccharate, stearate, succinate, tannate, tartrate, tosylate,trifluoroacetate and xinofoate salts. Suitable base salts are formedfrom bases which form non-toxic salts. Examples include, but are notlimited to, the ammonium, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine and zinc salts. Hemisalts of acids andbases may also be formed, for example, hemisulphate and hemicalciumsalts. For a review on suitable salts, see Handbook of PharmaceuticalSalts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH,2002), incorporated herein by reference.

Pharmaceutical compositions for use in the present disclosure typicallycomprise an effective amount of a compound and a suitablepharmaceutically acceptable carrier. The preparations may be prepared inany manner known per se, which usually involves mixing the at least onecompound according to the disclosure with the one or morepharmaceutically acceptable carriers, and, if desired, in combinationwith other pharmaceutical active compounds, when necessary under asepticconditions. Reference is again made to U.S. Pat. Nos. 6,372,778;6,369,086; 6,369,087 and 6,372,733 and the further references mentionedabove, as well as to the standard handbooks, such as the latest editionof Remington's Pharmaceutical Sciences.

Generally, for pharmaceutical use, the compounds may be formulated as apharmaceutical preparation comprising at least one compound of thepresent disclosure and at least one pharmaceutically acceptable carrier,diluent or excipient and/or adjuvant, and optionally one or more furtherpharmaceutically active compounds.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and may be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which may be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg, ofthe at least one compound of the disclosure, e.g. about 10, 25, 50, 100,200, 300, 400 or 500 mg per unit dosage.

The compounds can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. In certain embodiments, the compound is administeredby inhalation through the lungs.

The compound will generally be administered in an “effective amount”, bywhich is meant any amount of a compound that, upon suitableadministration, is sufficient to achieve the desired therapeutic orprophylactic effect in the subject to which it is administered. Usually,depending on the condition to be prevented or treated and the route ofadministration, such an effective amount will usually be between 0.01 to1000 mg per kilogram body weight of the patient per day, more oftenbetween 0.1 and 500 mg, such as between 1 and 250 mg, for example about5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of thepatient per day, which may be administered as a single daily dose,divided over one or more daily doses. The amount(s) to be administered,the route of administration and the further treatment regimen may bedetermined by the treating clinician, depending on factors such as theage, gender and general condition of the patient and the nature andseverity of the disease/symptoms to be treated. Reference is again madeto U.S. Pat. Nos. 6,372,778; 6,369,086; 6,369,087 and 6,372,733 and thefurther references mentioned above, as well as to the standardhandbooks, such as the latest edition of Remington's PharmaceuticalSciences.

Depending upon the manner of introduction, the compounds describedherein may be formulated in a variety of ways. Formulations containingone or more compounds can be prepared in various pharmaceutical forms,such as granules, tablets, capsules, suppositories, powders, controlledrelease formulations, suspensions, emulsions, creams, gels, ointments,salves, lotions, nanoparticles, aerosols and the like. Preferably, theseformulations are employed in solid dosage forms suitable for simple, andpreferably oral, administration of precise dosages. Solid dosage formsfor oral administration include, but are not limited to, tablets, softor hard gelatin or non-gelatin capsules, and caplets. However, liquiddosage forms, such as solutions, syrups, suspension, shakes, etc. canalso be utilized. In another embodiment, the formulation is administeredtopically. Suitable topical formulations include, but are not limitedto, lotions, ointments, creams, and gels. In a preferred embodiment, thetopical formulation is a gel. In another embodiment, the formulation isadministered intranasally.

In certain embodiments, the pharmaceutical composition comprises acompound disclosed herein and a propellant. In certain embodiments, anaerosolizing propellant is compressed air, ethanol, nitrogen, carbondioxide, nitrous oxide, hydrofluoroalkanes (HFAs),1,1,1,2,-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane orcombinations thereof.

In certain embodiments, the disclosure contemplates a pressurized orunpressurized container comprising a compound herein. In certainembodiments, the container is a manual pump spray, inhaler, meter-dosedinhaler, dry powder inhaler, nebulizer, vibrating mesh nebulizer, jetnebulizer, or ultrasonic wave nebulizer.

Formulations containing one or more of the compounds described hereinmay be prepared using a pharmaceutically acceptable carrier composed ofmaterials that are considered safe and effective and may be administeredto an individual without causing undesirable biological side effects orunwanted interactions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. As generally used herein “carrier” includes, but is notlimited to, diluents, binders, lubricants, disintegrators, fillers, pHmodifying agents, preservatives, antioxidants, solubility enhancers, andcoating compositions.

Carrier also includes all components of the coating composition whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release, extended release, and/or pulsatile releasedosage formulations may be prepared as described in standard referencessuch as “Pharmaceutical dosage form tablets”, eds. Liberman et al. (NewYork, Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et al., (Media, Pa.: Williams and Wilkins, 1995). Thesereferences provide information on carriers, materials, equipment andprocesses for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in thedrug-containing tablets, beads, granules or particles include, but arenot limited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers”,are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions.Non-limiting examples of anionic surfactants include sodium, potassium,ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates suchas sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, suchas sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, suchas sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates suchas sodium lauryl sulfate. Cationic surfactants include, but are notlimited to, quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include, but are not limited to, ethylene glycolmonostearate, propylene glycol myristate, glyceryl monostearate,glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucroseacylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylenemonolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butylether, Poloxamer® 401, stearoyl monoisopropanolamide, andpolyoxyethylene hydrogenated tallow amide. Examples of amphotericsurfactants include, but are not limited to, sodium N-dodecyl-β-alanine,sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaineand lauryl sulfobetaine.

If desired, the tablets, beads, granules, or particles may also containminor amount of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, or preservatives.

The concentration of the compound to carrier and/or other substances mayvary from about 0.5 to about 100 wt (weight percent). For oral use, thepharmaceutical formulation will generally contain from about 5 to about100% by weight of the active material. For other uses, thepharmaceutical formulation will generally have from about 0.5 to about50 wt. % of the active material.

The compositions described herein can be formulated for modified orcontrolled release. Examples of controlled release dosage forms include,but are not limited to, extended release dosage forms, delayed releasedosage forms, pulsatile release dosage forms, and combinations thereof.

The extended release formulations are generally prepared as diffusion orosmotic systems, for example, as described in “Remington—The science andpractice of pharmacy” (20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000). A diffusion system typically consists of twotypes of devices, a reservoir and a matrix, and is well known anddescribed in the art. The matrix devices are generally prepared bycompressing the drug with a slowly dissolving polymer carrier into atablet form. The three major types of materials used in the preparationof matrix devices are insoluble plastics, hydrophilic polymers, andfatty compounds. Plastic matrices include, but are not limited to,methyl acrylate-methyl methacrylate, polyvinyl chloride, andpolyethylene. Hydrophilic polymers include, but are not limited to,cellulosic polymers such as methyl and ethyl cellulose,hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCarbopol® 934, polyethylene oxides and mixtures thereof. Fatty compoundsinclude, but are not limited to, various waxes such as carnauba wax andglyceryl tristearate and wax-type substances including hydrogenatedcastor oil or hydrogenated vegetable oil, or mixtures thereof.

In certain preferred embodiments, the plastic material is apharmaceutically acceptable acrylic polymer, including but not limitedto, acrylic acid and methacrylic acid copolymers, methyl methacrylate,methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid),poly(methacrylic acid), methacrylic acid alkylamine copolymerpoly(methyl methacrylate), poly(methacrylic acid)(anhydride),polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), andglycidyl methacrylate copolymers.

In certain preferred embodiments, the acrylic polymer is comprised ofone or more ammonio methacrylate copolymers. Ammonio methacrylatecopolymers are well known in the art, and are described in NF XVII asfully polymerized copolymers of acrylic and methacrylic acid esters witha low content of quaternary ammonium groups.

In one preferred embodiment, the acrylic polymer is an acrylic resinlacquer such as that which is commercially available from Rohm Pharmaunder the tradename Eudragit® In further preferred embodiments, theacrylic polymer comprises a mixture of two acrylic resin lacquerscommercially available from Rohm Pharma under the tradenames Eudragit®RL30D and Eudragit® RS30D, respectively. Eudragit® RL30D and Eudragit®RS30D are copolymers of acrylic and methacrylic esters with a lowcontent of quaternary ammonium groups, the molar ratio of ammoniumgroups to the remaining neutral (meth)acrylic esters being 1:20 inEudragit® RL30D and 1:40 in Eudragit® RS30D. The mean molecular weightis about 150,000. Edragit® S-100 and Eudragit® L-100 are also preferred.The code designations RL (high permeability) and RS (low permeability)refer to the permeability properties of these agents. Eudragit® RL/RSmixtures are insoluble in water and in digestive fluids. However,multiparticulate systems formed to include the same are swellable andpermeable in aqueous solutions and digestive fluids.

The polymers described above such as Eudragit® RL/RS may be mixedtogether in any desired ratio in order to ultimately obtain asustained-release formulation having a desirable dissolution profile.Desirable sustained-release multiparticulate systems may be obtained,for instance, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit®RS, and 10% Eudragit® RL and 90% Eudragit® RS. One skilled in the artwill recognize that other acrylic polymers may also be used, such as,for example, Eudragit® L.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets andcapsules containing tablets, beads, or granules An immediaterelease portion can be added to the extended release system by means ofeither applying an immediate release layer on top of the extendedrelease core using a coating or compression process or in a multipleunit system such as a capsule containing extended and immediate releasebeads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In the congealingmethod, the drug is mixed with a wax material and either spray-congealedor congealed and screened and processed.

Delayed release formulations are created by coating a solid dosage formwith a polymer film, which is insoluble in the acidic environment of thestomach, and soluble in the neutral environment of the small intestine.

The delayed release dosage units can be prepared, for example, bycoating a drug or a drug-containing composition with a selected coatingmaterial. The drug-containing composition may be, e.g., a tablet forincorporation into a capsule, a tablet for use as an inner core in a“coated core” dosage form, or a plurality of drug-containing beads,particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and may be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Suitable coating materials for effecting delayed release include,but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, hydroxypropylmethyl cellulose phthalate, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate phthalate,cellulose acetate trimellitate and carboxymethylcellulose sodium;

acrylic acid polymers and copolymers, preferably formed from acrylicacid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate, and other methacrylic resinsthat are commercially available under the tradename Eudragit® (RohmPharma; Westerstadt, Germany), including Eudragit® L30D-55 and L100-55(soluble at pH 5.5 and above), Eudragit® L-100 (soluble at pH 6.0 andabove), Eudragit® S (soluble at pH 7.0 and above, as a result of ahigher degree of esterification), and Eudragits® NE, RL and RS(water-insoluble polymers having different degrees of permeability andexpandability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymer;enzymatically degradable polymers such as azo polymers, pectin,chitosan, amylose and guar gum; zein and shellac. Combinations ofdifferent coating materials may also be used. Multi-layer coatings usingdifferent polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 10 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethylcitrate, tributyl citrate, triethyl acetyl citrate, castor oil andacetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents arenonionic emulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 25 wt. % to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates may also be used. Pigmentssuch as titanium dioxide may also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), may also beadded to the coating composition.

The formulation can provide pulsatile delivery of the one or morecompounds. By “pulsatile” is meant that a plurality of drug doses arereleased at spaced apart intervals of time. Generally, upon ingestion ofthe dosage form, release of the initial dose is substantially immediate,i.e., the first drug release “pulse” occurs within about one hour ofingestion. This initial pulse is followed by a first time interval (lagtime) during which very little or no drug is released from the dosageform, after which a second dose is then released. Similarly, a secondnearly drug release-free interval between the second and third drugrelease pulses may be designed. The duration of the nearly drugrelease-free time interval will vary depending upon the dosage formdesign e.g., a twice daily dosing profile, a three times daily dosingprofile, etc. For dosage forms providing a twice daily dosage profile,the nearly drug release-free interval has a duration of approximately 3hours to 14 hours between the first and second dose. For dosage formsproviding a three times daily profile, the nearly drug release-freeinterval has a duration of approximately 2 hours to 8 hours between eachof the three doses.

In one embodiment, the pulsatile release profile is achieved with dosageforms that are closed and preferably sealed capsules housing at leasttwo drug-containing “dosage units” wherein each dosage unit within thecapsule provides a different drug release profile. Control of thedelayed release dosage unit(s) is accomplished by a controlled releasepolymer coating on the dosage unit, or by incorporation of the activeagent in a controlled release polymer matrix. Each dosage unit maycomprise a compressed or molded tablet, wherein each tablet within thecapsule provides a different drug release profile. For dosage formsmimicking a twice a day dosing profile, a first tablet releases drugsubstantially immediately following ingestion of the dosage form, whilea second tablet releases drug approximately 3 hours to less than 14hours following ingestion of the dosage form. For dosage forms mimickinga three times daily dosing profile, a first tablet releases drugsubstantially immediately following ingestion of the dosage form, asecond tablet releases drug approximately 3 hours to less than 10 hoursfollowing ingestion of the dosage form, and the third tablet releasesdrug at least 5 hours to approximately 18 hours following ingestion ofthe dosage form. It is possible that the dosage form includes more thanthree tablets. While the dosage form will not generally include morethan a third tablet, dosage forms housing more than three tablets can beutilized.

Alternatively, each dosage unit in the capsule may comprise a pluralityof drug-containing beads, granules or particles. As is known in the art,drug-containing “beads” refer to beads made with drug and one or moreexcipients or polymers. Drug-containing beads can be produced byapplying drug to an inert support, e.g., inert sugar beads coated withdrug or by creating a “core” comprising both drug and one or moreexcipients. As is also known, drug-containing “granules” and “particles”comprise drug particles that may or may not include one or moreadditional excipients or polymers. In contrast to drug-containing beads,granules and particles do not contain an inert support. Granulesgenerally comprise drug particles and require further processing.Generally, particles are smaller than granules, and are not furtherprocessed. Although beads, granules and particles may be formulated toprovide immediate release, beads and granules are generally employed toprovide delayed release.

In one embodiment, the compound is formulated for topicaladministration. Suitable topical dosage forms include lotions, creams,ointments, and gels. A “gel” is a semisolid system containing adispersion of the active agent, i.e., compound, in a liquid vehicle thatis rendered semisolid by the action of a thickening agent or polymericmaterial dissolved or suspended in the liquid vehicle. The liquid mayinclude a lipophilic component, an aqueous component or both. Someemulsions may be gels or otherwise include a gel component. Some gels,however, are not emulsions because they do not contain a homogenizedblend of immiscible components. Methods for preparing lotions, creams,ointments, and gels are well known in the art.

The compound described herein can be administered adjunctively withother active compounds. These compounds include, but are not limited to,analgesics, anti-inflammatory drugs, antipyretics, antidepressants,antiepileptics, antihi stamines, antimigraine drugs, antimuscarinics,anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators,anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics,electrolytes, gastro-intestinal drugs, muscle relaxants, nutritionalagents, vitamins, parasympathomimetics, stimulants, anorectics andanti-narcoleptics. “Adjunctive administration”, as used herein, meansthe compound can be administered in the same dosage form or in separatedosage forms with one or more other active agents.

Specific examples of compounds that can be adjunctively administeredwith the compounds include, but are not limited to, aceclofenac,acetaminophen, adomexetine, almotriptan, alprazolam, amantadine,amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine,amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine,beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone,bicifadine, bromocriptine, budesonide, buprenorphine, bupropion,buspirone, butorphanol, butriptyline, caffeine, carbamazepine,carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine,choline salicylate, citalopram, clomipramine, clonazepam, clonidine,clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine,corticosterone, cortisone, cyclobenzaprine, cyproheptadine,demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol,dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine,diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine,dihydroergotamine, dihydromorphine, dimetacrine, divalproxex,dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine,ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine,fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine,flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan,gabapentin, galantamine, gepirone, ginko bilboa, granisetron,haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone,hydroxyzine, ibuprofen, imipramine, indiplon, indomethacin, indoprofen,iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron,levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline,mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine,meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone,methamphetamine, methocarbamol, methyldopa, methylphenidate,methylsalicylate, methysergid(e), metoclopramide, mianserin,mifepristone, milnacipran, minaprine, mirtazapine, moclobemide,modafinil (an anti-narcoleptic), molindone, morphine, morphinehydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone,neurontin, nomifensine, nortriptyline, olanzapine, olsalazine,ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam,oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine,pemoline, pentazocine, pepsin, perphenazine, phenacetin,phendimetrazine, phenmetrazine, phenylbutazone, phenytoin,phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen,pizotyline, pramipexole, prednisolone, prednisone, pregabalin,propanolol, propizepine, propoxyphene, protriptyline, quazepam,quinupramine, reboxitine, reserpine, risperidone, ritanserin,rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, salsalate,sertraline, sibutramine, sildenafil, sulfasalazine, sulindac,sumatriptan, tacrine, temazepam, tetrabenozine, thiazides, thioridazine,thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin,toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine,trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid,venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone,zolmitriptan, zolpidem, zopiclone and isomers, salts, and combinationsthereof.

The additional active agent(s) can be formulated for immediate release,controlled release, or combinations thereof.

The present disclosure will now be described with reference to thefollowing non-limiting Examples.

EXPERIMENTAL

Synthesis

As illustrated in FIG. 4a , synthesis toward TFV disulfide conjugatesbegan with commercially available TFV (CombiBlocks) that were convertedto the bis-chloridate with excess oxalyl chloride and DMF indichloromethane (DCM). When catalytic DMF was used, the reaction stalleddue to the presence of the C-6 amino moiety on the purine ring systemwhich depletes the catalytic Vilsmeyer chlorinating agent and becomesconcomitantly protected as N-formimidine. This was resolved by adding1.2 equivalents of DMF to rapidly afford the formimidine protectedbis-chloridate that was subsequently treated with excessβ-mercaptoethanol to generate a mixture of bis- and mono-adducts ofcompound 6, which were not isolated. When compound 6 was stirred withaqueous saturated sodium bicarbonate for 30 minutes, the complexity ofthe mixture reduced to a single species whose m/z ratio was consistentwith compound 7. Acid-mediated hydrolysis of compound 7 affordedcompound 8 in moderate yield, whose structural assignment wasunambiguously characterized by HRMS, ¹H, ¹³C, and ³¹P NMR spectroscopy.A key step of the synthesis involved the construction of the criticaldisulfide linkage between compound 8 and hexadecanethiol. Unfortunately,this endeavor proved unsuccessful with a variety of oxidizing agentsincluding iodine, oxone, O₂, and H₂O₂. Cyclization of themercaptoethanol linker was observed when compound 8 reacted withmolecular iodine, and unidentifiable by-products or no appreciablereaction occurred with the latter oxidants. An alternative synthesis wasthen sought (as illustrated in FIG. 4b ). To this end, β-mercaptoethanolwas pre-oxidized with a handful of C₁₀-C₁₈ aliphatic thiols to producethe corresponding disulfide-bridged linkers 10a-e in moderate yields of34-45%. These compounds were crystalline solids with sharp meltingpoints (compound 10a is an oil) that could be cleanly purified by silicagel column chromatography. Alcohols 10a-e were subsequently coupled toTFV using the same DMF/oxalyl chloride methodology (shown above in FIG.4a ) to afford monoesters 11a-e in yields of 12-49% after hydrolysis ofthe remaining chloridate with water and deprotection of formimidine inwarm ethanol.

Despite the installation of a greasy hydrocarbon tail, the exposedphosphonic acid moiety retained significant polarity that initiallyplagued normal phase chromatographic purification of compounds 11a-e onsilica gel. Relatively polar solvent gradients of DCM/MeOH/NH₄OH(80:20:0.1) failed to move these compounds on aluminum-backed silica TLCplates and increasing the percentage of methanol compromised theintegrity of the silica and promoted streaking. However, when theconcentration of ammonium hydroxide was raised from 0.1 to 1-3% (v/v),appreciable movement was observed by TLC and these conditions weresuccessfully applied to column chromatography to furnish compounds 11a-eas their ammonium salts, as determined by elemental analysis.

With monoesters 11a-e in hand, the corresponding bis-disulfides 12a-e(shown in FIG. 5) were prepared employing the same protocol and usingtwo equivalents of compounds 10a-e during coupling to TFV instead ofone. All five transformations occurred as predicated to afford compounds12a-e in 30-40% yield after purification without incident.

A series of mixed phosphonodiesters (compounds 13-21) were synthesizedand are also displayed in FIG. 5. The synthesis of these compounds wasdone using compound 10d as the coupling lipid and variousalcohols/amines to quench the remaining phosphonochloridate in step 3 ofthe reaction scheme shown in FIG. 4b , rather than water. Note thatcompounds 13-21 were prepared as an inconsequential mixture ofdiastereomers and were not separated.

As illustrated in FIG. 6, conjugates 21-25c, 26, and 27 were alsoprepared. Hexadecanethiol was oxidized with thiols 21-25a in thepresence of iodine to furnish lipids 21-25b as low-melting point solidsthat were purified via column chromatography. Compounds 21-25b were thencoupled to TFV with oxalyl chloride and DMF to afford the correspondingconjugates 21-25c after formimidine deprotection and purification onsilica gel using a DCM:MeOH:NH₄OH gradient. Both compounds 26 and 27were purified on a C₁₈ reverse phase column and were isolated as freeacids following lyophilization. The structures of compounds 21-25c, 26,and 27 are presented in Table 3.

Antiviral Activity

Compounds synthesized were evaluated against HIV-infected PBMCs andantiviral activity was assessed by measuring reverse transcriptase incell supernatants. Compounds 11a-e, 12a-e, and 13 were assayed againstHBV and qPCR was used to quantify viral DNA following incubation inHepG2.2.15 cells after 6 days. Table 1 details the antiviral activity ofconjugates 11a-e with modifications to the lipid tail.

TABLE 1 HIV-1 and HBV Activity of Lysogenic Phospholipids 11a-e Comparedto TDF, CMX-157, and DTE-TFV HIV-1 HBV EC₅₀ ^(b) CC₅₀ ^(c) TI EC₅₀ ^(b)CC₅₀ ^(c) TI Compound (PBMCs) (PBMCs) (CC₅₀/EC₅₀) (HepG2) (HepG2)(CC₅₀/EC₅₀) TDF 0.0046 43.7 9,500 0.34 64.5 190 TFV 0.319 >100 >300 — —— 8 18.6 >100.0 >5.38 41.9 >100.0 >2.4 DTE-TFV 1.61 >100.0 >62.111.6 >100.0 >8.6 11a 0.085 >50.0 >590 >50.0 >50.0 >1 11b 0.0030 25.08,300 1.67 >50.0 >30 11c 0.00050 14.0 28,000 0.444 >25 >56 11d 0.0006514.3 22,000 0.020 >25 >1250 CMX-157 0.02 >100.0 >5000 — — — 11e 0.000606.36 11,000 0.505 >50 >50 Data represent an average of triplicateexperiments. ^(b)EC₅₀, effective concentration (in μM) required toinhibit HIV-1 or HBV by 50%. ^(c)CC₅₀, effective concentration (in μM)required to reduce the viability of uninfected cells by 50%.

These compounds resemble lysogenic phospholipids characterized by ananionic phosphate head group and a single aliphatic tail. Thisstructural motif confers a conical-like shape that facilitates faciletranslocation between the inner and outer leaflets of the plasmamembrane and procures detergent-like properties that disrupts lipidbi-layers. As shown in Table 1, compound 11a possesses the shortestlipid (C₁₀) and is 18-fold less potent than TDF (85 nM vs. 4.6 nM) witha relatively poor therapeutic index (TI) of 590 at the EC₅₀. Extendingthe length of the alkyl chain by two carbon atoms results in compound11b whose HIV-1 activity (3.0 nM) is comparable to TDF and seven-foldmore active than CMX-157 (20 nM). Maximum antiviral activity wasobtained for conjugates 11c-e with alkyl chain lengths ranging from14-18 carbon atoms, respectively. Compounds 11c-e exhibit sub-nanomolarEC₅₀ values of ˜0.5 nM that outrivals TDF and CMX-157 and compound 11dboasts a TI₅₀ that exceeds 20,000 whereas that of compound 11capproaches 30,000. This broad therapeutic window is sustained even atthe EC₉₀ for both compounds 11c and 11d and is nearly an order ofmagnitude wider than the TI₉₀ of TDF (11,000 vs. 1,300—data not shown).Note that compounds 11c-e possess linker lengths of 19, 20, and 21atoms, respectively, when the mercaptoethanol bridge is taken intoaccount. Table 1 also reveals that increasing chain length is associatedwith a concomitant increase in cytotoxicity, a phenomenon that has beenwell-documented for a variety of surfactants in numerous aquaticorganisms.

With respect to HBV activity, all conjugates in Table 1 demonstratedrather unremarkable activity with the exception of compounds 11d whosepotency (20 nM) and TI (1250) bested that of TDF which further supportsthe advantage of reduction-sensitive lipids over carbonate prodrugstrategies.

In addition to compounds 11a-e, DTE-TFV and 8 were also assessed in thisassay and were found to be 5- and 58-fold less active than TFV againstHIV-1, respectively. The dramatic potency loss observed for compound 8is interesting given that this compound is predicated to readily undergointramolecular cyclization to release the bound nucleoside in vivo.Without wishing to be limited to any particular theory, it is suspectedthat the β-mercaptoethanol linker resists cleavage followingintracellular delivery and obstructs the phosphorylation of the parentnucleoside to the active diphosphate.

In contrast to compounds 11a-e, compounds 12a-e resemble conventionalphospholipids with two aliphatic tails that assume a cylindrical shapeand do not readily traverse the plasma membrane on their own accord.Phosphonodiesters in Table 2 proved to be less potent than compounds11a-e.

TABLE 2 HIV-1 and HBV Activity of Bis-disulfide Conjugates 12a-e HIV-1HBV EC₅₀ ^(b) CC₅₀ ^(c) TI EC₅₀ ^(b) CC₅₀ ^(c) TI Compound (PBMCs)(PBMCs) (CC₅₀/EC₅₀) (HepG2) (HepG2) (CC₅₀/EC₅₀) TDF 0.0046 43.7 9,5000.34 29.2 86 12a 0.349 >100.0 >287 >100 >100.0 >1 12b1.18 >100.0 >85.0 >100 >100.0 >1 12c 0.189 >100.0 >529 >100 >100.0 >112d 6.32 >25 4.0 >25 >25 >1 12e 0.331 >25 >76 >25 >25 >1 Data representan average of triplicate experiments. ^(b)EC₅₀, effective concentration(in μM) required to inhibit HIV-1 or HBV by 50%. ^(c)CC₅₀, effectiveconcentration (in μM) required to reduce the viability of uninfectedcells by 50%.

Conjugates 12a and 12e achieved potencies comparable to TFV againstHIV-1 whereas compound 12c was the only compound to demonstrate atwo-fold increase in activity when compared to the parent nucleoside.

Tested compounds were relatively inactive against HBV relative to TDF.The translocation of compounds 12a-e from the outer leaflet to innerleaflet of the plasma membrane is required in order for TFV to blockadereverse transcriptase located within the cytosol. If it is assumed thatreduction is facile upon entry and that compounds 11a-e and 12a-einteract with the cellular machinery following cleavage of the linker,then the results presented in Table 2 strongly implicate membranetranslocation as the rate-limiting step governing the EC₅₀ value ofcompounds 12a-e. It is therefore interesting that compounds 12a, c and eand TFV (Table 2) all have strikingly similar HIV-1 activity despitetheir disparate alkyl chain lengths and in the case of TFV, no chain atall.

Conjugates 22-24c (as illustrated in FIG. 6 and Table 3) were evaluatedagainst HIV-1 infected PBMCs and chronically HBV-infected hepatocytes.TFV and TDF were also evaluated and serve as reference compounds toassess the efficacy of this prodrug strategy. Thiols 22-24a werejudiciously selected based on commercial availability and theirpropensity for cyclization when the hydroxyl moiety bears a potentialleaving group (i.e. TFV). Note that compound 23a is expected to undergoo-thioquinone methide formation rather than intramolecular cyclization.As shown in Table 3, both compounds 22c and 24c have similar HIV-1activity to conjugate 21c (˜0.5 nM). However, compound 22c issignificantly less cytotoxic than compounds 21c and 24c (CC₅₀>50 μM). Anaccurate determination of the CC₅₀ for compound 22c was not possible dueto precipitation at concentrations higher than 50 μM. Nonetheless, theattenuated toxicity of compound 22c combined with its potent antiviralactivity achieves a TI₅₀ in excess of 100,000 providing a relativelynon-toxic disulfide bearing lipid prodrug of TFV.

TABLE 3 HIV-1 and HBV Activity of Conjugates 21-25c, 26, and 27 Comparedto TFV and TDF

HIV-1 HBV EC₅₀ ^(b) CC₅₀ ^(c) TI EC₅₀ ^(b) CC₅₀ ^(c) TI Id. Structure(PBMCs) (PBMCs) (CC₅₀/EC₅₀) (HepG2) (HepG2) (CC₅₀/EC₅₀) TFV

0.320 >100 >300 — — — TDF

0.0045 44.0 9,500 0.34 64.5 190 21c

0.00065 14.3 22,000 0.020 >25 >1200 22c

<0.0005 >50 >100,000 0.248 >50 >200 23c

0.0229 17.2 751 4.48 17.5 3.9 24c

<0.0005 15.9 >31,800 0.152 32.5 214 25c

0.007 >50 >7000 1.05 >50 >47 26

18.6 >100 >5.38 41.9 >100 >2 27

5.13 >100 >18 >100 >100 >1 Data represent an average of triplicateexperiments. ^(b)EC₅₀, effective concentration (in μM) required toinhibit HIV-1 or HBV by 50%. ^(c)CC₅₀, effective concentration (in μM)required to reduce the viability of uninfected cells by 50%.Stability Studies

In order to be considered clinical candidates for oral delivery,conjugates 11b-e must demonstrate sufficient hydrolytic, nucleophilic,and plasma stability. Bis(DTE)-conjugates have dismal reported plasmastability profile of (t_(1/2)<5 min). Compound 11c and 11d were selectedas model compounds and subjected to various media including human serum,PBS buffer (pH 7.4), Dulbecco's Modified Eagle Medium (DMEM), andcarbonate/bicarbonate buffer (pH 9). Aliquots of each sample wereanalyzed by LC-MS at varying times points over the course of 2 hourswith a final time point at 24 to assess decomposition.

Both compounds 11c and 11d demonstrated robust stability in human plasmawith a half-life of >24 h. In contrast, DTE-TFV readily degraded in thepresence of human plasma (t_(1/2)=34 min) which is in agreement withprevious findings.

Experiments were performed to see how compounds how compounds 11c and11d would fare in the presence of base (pH 9), PBS (pH 7.4), and uponexposure to nucleophilic media (DMEM). Preliminary experiments withcompound 11d and DTE-TFV in PBS initially revealed that compound 11drapidly decomposed in PBS solution with t_(1/2)<20 min while DTE-TFVboasted a half-life of 3.5 h. Poor PBS stability was also observed forother alkoxyalkyl conjugates in PBS, but not for less hydrophobicconjugates. In light of these observations, it was suspected thatchemical decomposition was not the operative mechanism behind thisphenomenon in PBS, but rather physical adsorption of the lipid to theglass surface was responsible for abstracting analyte from the solution.To this end, the stability of hexadecyloxypropyl2′-deoxy-2′-fluorouridine, a non-labile alkoxyalkyl lipid conjugate of2′-deoxy-2′-fluorouridine in PBS buffer using Pyrex, Kimax, andsilanized glassware was assessed. These experiments revealed that bothPyrex and silanized glass encourage substantial lipid adsorption andgive rise to a rapid (but artificial) decomposition profile forlipid-bound nucleosides (data not shown). Interestingly, Kimax glasswaredid not produce this effect despite its near identical composition toPyrex. This prompted re-assessment of the PBS stability of the disulfidelipid conjugates in Kimax glassware. Note that all stability experimentswere performed in Kimax glassware using hexadecyloxypropyl2′-deoxy-2′-fluorouridine as an internal standard. With theseconditions, both compounds 11c and 11d exhibited similar stabilities atpH 9, PBS, and DMEM. Neither compound 11c nor compound 11d exhibit ahalf-life of >2 h in these media which is in stark contrast to theirstability in human serum (>24 h). It is believed that the precipitationof compounds 11d and 11c from the media is responsible for the poorhalf-life of these compounds in PBS, carbonate buffer, and DMEM. This isconsistent with the observation that the C₁₄ chain of compound 11cprocures a near two-fold stability increase over the C₁₆ linker ofcompound 11d in all examined media (with the exception of human serum).It is plausible that the proteins present in human serum provideadditional Van der Waals contacts and support micelle formation tofacilitate dissolution. Taken together, the inherent plasma stability ofcompounds 11c and 11d illuminates the possibility for selectiveintracellular delivery that may reduce systemic exposure of TFV or otherbound cargo.

As a Tool to Probe Enzymatic Cleavage.

A subset of prodrug strategies relies on the presence of specifichydrolases such as phospholipase C or cathepsin A to sever the P-O orP-N linkages between prodrug and nucleoside. Unfortunately, little isknown about the substrate specificity of these enzymes. Seminal reportsby Kelly et al. concluded 5′-nucleotide phosphodiesterase preferentiallycleaves aryl esters over aliphatic and benzyl derivatives. However, asystematic analysis of various ester derivatives has not been examinedin PBMCs. This was seen as an opportunity to indirectly probe theenzymatic machinery responsible for the hydrolysis of TFVphosphonomonoesters in PBMCs using the reduction-sensitive lipids as adelivery vehicle.

Conjugates 13-21 were prepared using hexadecyldisulfanylethanol (HDE) asthe lipid of choice. HDE ensures thorough cellular permeation andrelatively rapid intracellular cleavage to expose the correspondingphosphonomonoester which is then subsequently hydrolyzed in arate-limiting step to TFV. The rate of hydrolysis is dependent on thenature of the alkyl or aryl ester and therefore governs the HIV-1activity of compounds 13-21. None of these compounds (13-21) exhibitedantiviral activity comparable to compounds 11c-e due to the additionalcleavage step (Table 4).

TABLE 4 HIV-1 Activity of Lipid Conjugates 13-21 EC₅₀ ^(b) CC₅₀ ^(c)TI(CC₅₀/ Compound R- (PBMCs) (PBMCs) EC₅₀) TDF N.A 0.0046 43.7 9,500 TFVN.A 0.319 >100.0 >300 13 MeO— 0.026 38.2 1,500 14 EtO— 0.349 36.2 104 15PrO— 0.216 10.6 49.0 16 n-BuO— 0.19 27.9 150 17 s-BuO— 0.049 23.7 480 18C(CH₃)₃CH₂O— 0.14 >100.0 >700 19 PhCH₂O— 0.018 41.1 2,300 20 PhNH— 0.00534.2 7,000 21 C1₈H₃₇O— 0.911 >50.0 >50 Data represent an average oftriplicate experiments. ^(b)EC₅₀, effective concentration (in μM)required to inhibit HIV-1 or HBV by 50%. ^(c) CC₅₀, effectiveconcentration (in μM) required to reduce the viability of uninfectedcells by 50%.

Successively increasing the chain length of the alkyl ester from methylto n-Bu resulted in significant potency loss with compound 13 being themost active (26 nM) of the linear series (compounds 13-15, 16 and 21).When compound 13 is omitted, HIV-1 activity concomitantly increased withchain length from ethyl to n-Bu. Data suggests that cleavage of methylmonoester 13 in human PBMCs is superior to the corresponding ethyl,propyl, and n-butyl esters (14, 15, 16, respectively) and thatconversion to TFV increases with chain length for this series. Note thatthe antiviral activity of compounds 13-21 directly corresponds to TFVand thus provides an indirect readout for the concentration of the freenucleoside.

Longer alkyl chains impose significant phospholipid character with theHDE linker and become “stuck” in the plasma membrane—a phenomenonobserved with conjugates 12a-e. The activity of compound 21 (0.911 μM)is likely plagued by the same phenomenon and obscures the relationshipbetween the EC₅₀ and enzymatic cleavage of the octyl ester. Fortunately,the remaining esters in Table 4 are sufficiently smaller than the HDElinker and do not exhibit this behavior.

Conjugates 16, and 17-18 reveal that a moderate increase in stericencumbrance around the phosphonate center correlates with an increase inantiviral activity. The s-Bu ester 17 is superior to its n-Bu analogue16 by nearly four-fold. The neopentyl derivative 18 had unimpressiveactivity of 0.14 μM although the corresponding n-pentyl ester was notsynthesized for comparison.

Aryl phosphonoamidate 20 and benzyl ester 19 are the most potentcompounds in Table 4 (5 nM and 18 nM, respectively) which is consistentwith the notion that aryl and benzyl esters are superior substrates overtheir alkyl counterparts.

The invention claimed is:
 1. A method of treating a viral infectionselected from the group consisting of human immunodeficiency virus (HIV)infection and hepatitis B virus (HBV) infection, comprisingadministering to a subject in need thereof an effective amount of acompound of Formula I,

or a salt thereof, wherein, W is

Z is O, S, or Se; Y is O, S, or NH; T is an aryl or alkyl linking group;R¹ is a saturated hydrocarbon chain having 6 or more carbon atoms; R² isR¹SST-, hydrogen, alkyl, aryl, phenyl, 4-fluorophenyl, 4-chlorophenyl,4-bromophenyl, naphthyl, or heterocyclyl, wherein R² is optionallysubstituted with one or more, the same or different, R¹⁰; R¹⁰ isdeuterium, alkyl, alkenyl, alkynyl, alkanoyl, halogen, nitro, cyano,hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkylsulfonyl, arylsulfonyl, benzyl, benzoyl, carbocyclyl, aryl, orheterocyclyl, wherein R¹⁰ is optionally substituted with one or more,the same or different, R¹¹; and R¹¹ is deuterium, halogen, nitro, cyano,hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl,acetoxy, methylamino, ethylamino, dimethylamino, diethylamino,N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl,N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl,methylthio, ethylthio, methyl sulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,N-methyl-N-ethylsulfamoyl, benzyl, benzoyl, carbocyclyl, aryl, orheterocyclyl.
 2. The method of claim 1, wherein T is a C₂ to C₆ alkyl.3. The method of claim 1, wherein Z and Y are each O.
 4. The method ofclaim 1, wherein R¹ is a C₆ to C₂₀ saturated hydrocarbon chain.
 5. Themethod of claim 1, wherein R² is R¹SST-, hydrogen, alkyl, aryl orphenyl.
 6. The method of claim 1, wherein R² is hydrogen, methyl, oralkyl.
 7. The method of claim 1, wherein the compound is selected fromthe following compounds as free acids or salts thereof:


8. The method of claim 1 wherein the subject is at risk of, exhibitingsymptoms of, suffering from, or diagnosed with the viral infection. 9.The method of claim 1 wherein the compound is administered incombination with another antiviral agent.
 10. The method of claim 1,wherein T is —(CH₂)_(n)—, wherein n is 2, 3, 4, 5, or
 6. 11. The methodof claim 10, wherein n is
 2. 12. The method of claim 10, wherein n is 4or
 5. 13. The method of claim 1, wherein T is —C₆H₄—, —CH₂—C₆H₄—,—C₆H₄—CH₂—, or —CH₂—C₆H₄—CH₂—.
 14. The method of claim 1, wherein theviral infection is HIV infection.
 15. The method of claim 1, wherein theviral infection is HBV infection.